Methods and compositions for the inhibition of gene expression

ABSTRACT

The present invention relates to methods and compositions for the inhibition of gene expression. In particular, the present invention provides oligonucleotide-based therapeutics for the inhibition of oncogenes involved in cancers.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for theinhibition of gene expression. In particular, the present inventionprovides oligonucleotide-based therapeutics for the inhibition ofoncogenes involved in cancers.

BACKGROUND OF THE INVENTION

Oncogenes have become the central concept in understanding cancerbiology and may provide valuable targets for therapeutic drugs. Alloncogenes and their products operate inside the cell. This makesprotein-based drugs ineffective since their specificity involvesligand-receptor recognition.

Antisense oligodeoxyribonucleotides (oligonucleotides) are underinvestigation of therapeutic compound for specifically targetingoncogenes (Wickstrom, E. (ed). Prospects for antisense nucleic acidtherapy of cancer and Aids. New York: Wiley-Liss, Inc. 1991; Murray, J.A. H. (ed). Antisense RNA and DNA New York: Wiley-Liss, Inc. 1992).Antisense drugs are modified synthetic oligonucleotides that work byinterfering with ribosomal translation of the target mRNA. The antisensedrugs developed thus far destroy the targeted mRNA by binding to it andtriggering ribonuclease H(RNase H) degradation of mRNA. Oligonucleotideshave a half-life of about 20 minutes and they are therefore rapidlydegraded in most cells (Fisher, T. L. et al., Nucleic Acids Res.21:3857-3865 (1993)). To increase the stability of oligonucleotides,they are often chemically modified, e.g., they are protected by a sulfurreplacing one of the phosphate oxygens in the backbone(phosphorothioate) (Milligan, J. F. et al., J. Med. Chem. 36:1923-1937(1993); Wagner, R. W. et al., Science 260:1510-1513 (1993)). However,this modification can only slow the degradation of antisense andtherefore large dosages of antisense drug are required to be effective.

Despite the optimism surrounding the use of antisense therapies, thereare a number of serious problems with the use of antisense drugs such asdifficulty in getting a sufficient amount of antisense into the cell,non-sequence-specific effects, toxicity due to the large amount ofsulfur containing phosphothioates oligonucleotides and their inabilityto get into their target cells, and high cost due to continuous deliveryof large doses. An additional problem with antisense drugs has beentheir nonspecific activities.

What is needed are additional non-protein based cancer therapeutics thattarget oncogenes. Therapeutics that are effective in low doses and thatare non-toxic to the subject are particularly needed.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for theinhibition of gene expression. In particular, the present inventionprovides oligonucleotide-based therapeutics for the inhibition ofoncogenes involved in cancers.

In some embodiments, the present invention provides a compositioncomprising a first oligonucleotide that hybridizes under physiologicalconditions to the promoter region of a bcl-3 gene (e.g., SEQ ID NOs: 3,7, 8 or 9). In some embodiments, at least one of the cytosine bases inthe first oligonucleotide is 5-methylcytosine. In some embodiments, allof the cytosine bases in the first oligonucleotide are 5-methylcytosine.In some preferred embodiments, the hybridization of the firstoligonucleotide to the promoter region of a bcl-2 gene inhibitsexpression of the bcl-2 gene. In some embodiments, the bcl-2 gene is ona chromosome of a cell, and the hybridization of the firstoligonucleotide to the promoter region of bcl-2 gene reducesproliferation of the cell. In some embodiments, the composition furthercomprises a second oligonucleotide. In some embodiments, the at leastone (e.g. all) of the cytosines in the second oligonucleotide are5-methylcytosine. In some embodiments, the second oligonucleotidecomprises SEQ ID NOs: 3, 7, 8 or 9, and is different than the firstoligonucleotide (e.g., if the second oligonucleotide has the sequence ofSEQ ID NO:3, the first oligonucleotide has a sequence other than SEQ IDNO:3, etc.). In some embodiments, the second oligonucleotide hybridizesto a promoter region of a second gene, wherein the second gene is notbcl-2. In some embodiments, the second gene is an oncogene (e.g.,c-ki-Ras, c-Ha-Ras, c-myc, Her-2, or TGF-α).

In other embodiments, the present invention provides a compositioncomprising an oligonucleotide that hybridizes to a promoter region of abcl-2 gene at a position comprising between nucleotides 1-40 of SEQ IDNO:1, between nucleotides 161-350 of SEQ ID NO:1, between nucleotides401-590 of SEQ ID NO:1 or between nucleotides 1002-1260 of SEQ ID NO: b1.

In yet other embodiments, the present invention provides a method,comprising: providing an oligonucleotide (e.g., SEQ ID NOs: 3, 7, 8, or9); and a cell capable of proliferation, and comprising a bcl-2 genecapable of being expressed; and introducing the oligonucleotide to thecell. In some embodiments, the introducing results in the reduction ofproliferation of the cell. In certain embodiments, the introducingresults in inhibition of expression of the bcl-2 gene. In someembodiments, the cell is a cancer cell. In other embodiments, the cellis in a host animal (e.g., a non-human mammal or a human). In someembodiments, the oligonucleotide is introduced to the host animal at adosage of between 0.01 μg to 100 g, and preferably at a dosage ofbetween 1 mg to 100 mg per kg of body weight. In some embodiments, theoligonucleotide is introduced to the host animal one or more times perday. In other embodiments, the oligonucleotide is introduced to the hostanimal continuously. In still further embodiments, the cell is in cellculture. In some embodiments, the method further comprises the step ofintroducing a test compound to the cell. In some embodiments, the testcompound is a known chemotherapy agent. In some embodiments, the canceris pancreatic cancer, colon cancer, breast cancer, bladder cancer, lungcancer, leukemia, prostate, lymphoma, ovarian, or melanoma. In someembodiments, at least one (e.g., all) of the cytosine bases in theoligonucleotide are 5-methylcytosine.

The present invention further provides a method, comprising: providingan oligonucleotide that hybridizes to the promoter region of a bcl-2gene; and a cell comprising a bcl-2 gene; and introducing theoligonucleotide to the cell. In some embodiments, the oligonucleotidecomprises at least one CG dinucleotide pair, wherein at least one of thecytosine bases in the CG dinucleotide pair is 5-methylcytosine. In someembodiments, the oligonucleotide is completely complementary to thepromoter region of the bcl-2 gene. In other embodiments, theoligonucleotide is partially complementary to the promoter region of thebcl-2 gene. For example, in certain embodiments, the oligonucleotidecontains one mismatch to the promoter region of the bcl-2 gene. In somepreferred embodiments, the oligonucleotide is complementary only to thepromoter region of the bcl-2 gene and is not completely complementary toother regions of the human genome. In some embodiments, theoligonucleotide is between 10 nucleotides and 60, and preferably between15 and 35 nucleotides in length.

In some embodiments, the method further provides a drug delivery system.In some embodiments, the drug delivery system comprises a liposome(e.g., a liposome comprising a neutral lipid or a lipid like compound).In some embodiments, the drug delivery system comprises a cell targetingcomponent (e.g., a ligand or ligand like molecule for a cell surfacereceptor or a nuclear receptor). In certain embodiments, the drugdelivery system is for use in vivo, and the oligonucleotide and theliposome are present in the ratio of from 2:1 to 1:3/1 μg to 100 mg perkg body weight.

In still further embodiments, the present invention provides acomposition comprising an oligonucleotide that hybridizes underphysiological conditions to the promoter region of a bcl-2 gene. In someembodiments, the oligonucleotide comprises at least one CG dinucleotidepair, wherein at least one of the cytosine bases in the CG dinucleotidepair is 5-methylcytosine. In some embodiments, the oligonucleotide iscompletely complementary to the promoter region of the bcl-2 gene. Inother embodiments, the oligonucleotide is partially complementary to thepromoter region of the bcl-2 gene. For example, in certain embodiments,the oligonucleotide contains one mismatch to the promoter region of thebcl-2 gene. In some preferred embodiments, the oligonucleotide iscomplementary only to the promoter region of the bcl-2 gene and is notcompletely complementary to other regions of the human genome. In someembodiments, the oligonucleotide is between 10 nucleotides and 60, andpreferably between 15 and 35 nucleotides in length.

The present invention further provides a composition comprising anoligonucleotide that hybridizes under physiological conditions to thepromoter region of a bcl-2 gene under conditions such that expression ofthe bcl-2 gene is inhibited.

The present invention additionally provides a composition comprising anoligonucleotide that hybridizes under physiological conditions to thepromoter region of a bcl-2 gene located on a chromosome of a cell underconditions such that proliferation of the cell is reduced.

The present invention also provides a composition comprising a firstoligonucleotide that hybridizes under physiological conditions to thepromoter region of a bcl-2 gene, the oligonucleotide comprising at leaston CG dinucleotide pair, wherein at least one of the cytosine bases inthe CG dinucleotide pair comprises 5-methylcytosine; and a secondoligonucleotide, the second oligonucleotide comprising at least on CGdinucleotide pair, wherein at least one of the cytosine bases in the CGdinucleotide pair comprises 5-methylcytosine.

In certain embodiments, the present invention provides a kit comprisingan oligonucleotide that hybridizes under physiological conditions to thepromoter region of a bcl-2 gene, the oligonucleotide comprising at leaston CG dinucleotide pair, wherein at least one of the cytosine bases inthe CG dinucleotide pair is 5-methylcytosine; and instructions for usingthe kit for reducing proliferation of a cell comprising a bcl-2 gene ona chromosome of the cell or inhibiting gene expression. In someembodiments, the composition in the kit are used for treating cancer ina subject and the instructions comprise instructions for using the kitto treat cancer in the subject. In some embodiments, the instructionsare instructions required by the U.S. Food and Drug Agency for labelingof pharmaceuticals.

The present invention also provides a method, comprising: providing abiological sample from a subject diagnosed with a cancer; and reagentsfor detecting the present or absence of expression of a oncogene in thesample; and detecting the presence or absence of expression of anoncogene in the sample; introducing an oligonucleotide that hybridizesunder physiological conditions to the promoter region of an oncogeneexpressed in the biological sample, the oligonucleotide comprising atleast on CG dinucleotide pair, wherein at least one of the cytosinebases in the CG dinucleotide pair is 5-methylcytosine to the subject.

The present invention additionally provides a method of inhibiting theexpression of a gene in a subject (e.g., for the treatment of cancer orother hyperproliferative disorders) comprising providing anoligonucleotide that hybridizes under physiological conditions to thepromoter region of a gene involved in cancer or a hyperproliferativedisorder expressed in the biological sample, the oligonucleotidecomprising at least on CG dinucleotide pair, wherein at least one of thecytosine bases in the CG dinucleotide pair comprises 5-methylcytosine;and administering the oligonucleotide to the subject under conditionssuch that expression of the gene is inhibited. In some embodiments, thesubject is a human.

In yet further embodiments, the present invention provides a method ofscreening compounds comprising providing a cell comprising a suspectedoncogene; and an oligonucleotide that hybridizes to the promoter regionof the gene; and introducing the oligonucleotide to the cell; anddetermining if proliferation of the cell is inhibited in the presence ofthe oligonucleotide relative to the absence of the oligonucleotide. Insome embodiments, the cell is in culture (e.g., a cancer cell line). Inother embodiments, the cell is in a host animal (e.g., a non-humanmammal). In some embodiments, the method is a high-throughput screeningmethod.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleic acid sequence of the bcl-2 gene (SEQ ID NO:1).

FIG. 2 shows the sequences of antigenes to bcl-2 used in someembodiments of the present invention. X refers to a methylated Cnucleotide.

FIG. 3 shows the nucleic acid sequence of the c-erbB-2 (Her-2) gene (SEQID NO:29).

FIG. 4 shows the sequences of antigenes to c-erbB-2 used in someembodiments of the present invention. X refers to a methylated Cnucleotide.

FIG. 5 shows the nucleic acid sequence of the c-ki-Ras gene (SEQ IDNO:46).

FIG. 6 shows the sequences of antigenes to c-ki-Ras used in someembodiments of the present invention. X refers to a methylated Cnucleotide.

FIG. 7 shows the nucleic acid sequence of the c-Ha-Ras gene (SEQ IDNO:66).

FIG. 8 shows the sequences of antigenes to c-Ha-Ras used in someembodiments of the present invention. X refers to a methylated Cnucleotide.

FIG. 9 shows the nucleic acid sequence of the c-myc gene (SEQ IDNO:108).

FIG. 10 shows the sequences of antigenes to c-myc used in someembodiments of the present invention. X refers to a methylated Cnucleotide.

FIG. 11 shows the nucleic acid sequence of the TGF-α gene (SEQ IDNO:131).

FIG. 12 shows the sequences of antigenes to TGF-α used in someembodiments of the present invention. X refers to a methylated Cnucleotide.

FIG. 13 shows the inhibition of expression of cell growth by antigenesto c-ki-Ras used in some embodiments of the present invention FIG. 14shows the inhibition of expression of cell growth by antigenes to bcl-2used in some embodiments of the present invention.

FIG. 15 shows the inhibition of expression of cell growth by antigenesto c-erb-2 used in some embodiments of the present invention.

FIG. 16 shows the inhibition of expression of cell growth by antigenesto c-Ha-Ras used in some embodiments of the present invention.

FIG. 17 shows the inhibition of expression of cell growth by antigenesto c-myc used in some embodiments of the present invention.

FIG. 18 shows the inhibition of expression of cell growth by antigenesto TGF-α used in some embodiments of the present invention.

FIG. 19 shows the dose response curve of inhibition of expression ofcell growth by antigenes to c-ki-Ras.

FIG. 20 shows the dose response curve of inhibition of expression ofcell growth of FSCCL cells (A) and MCF-7 cells (B) by antigenes tobcl-2.

FIG. 21 shows the dose response curve of inhibition of expression ofcell growth by antigenes to c-erb-2.

FIG. 22 shows the dose response curve of inhibition of expression ofcell growth by antigenes to c-Ha-Ras used.

FIG. 23 shows the dose response curve of inhibition of expression ofcell growth by antigenes to c-myc.

FIG. 24 shows the dose response curve of inhibition of expression ofcell growth of T47D cells (A) and MDA-MB-231 cells (B) by antigenes toTGF-α.

FIG. 25 shows exemplary variants of antigenes to c-ki-Ras.

FIG. 26 shows exemplary variants of antigenes to bcl-2.

FIG. 27 shows exemplary variants of antigenes to c-erb-2.

FIG. 28 shows exemplary variants of antigenes to c-ha-ras.

FIG. 29 shows exemplary variants of antigenes to c-myc.

FIG. 30 shows exemplary variants of antigenes to TGF-α.

FIG. 31 shows inhibition of lymphoma cells by non-methylatedoligonucleotides targeted toward Bcl-2.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “under conditions such that expression of saidgene is inhibited” refers to conditions where an oligonucleotide of thepresent invention hybridizes to a gene (e.g., the promoter region of thegene) and inhibits transcription of the gene by at least 10%, preferablyat least 25%, even more preferably at least 50%, and still morepreferably at least 90% relative to the level of transcription in theabsence of the oligonucleotide. The present invention is not limited tothe inhibition of expression of a particular gene. Exemplary genesinclude, but are not limited to, c-ki-Ras, c-Ha-ras, c-myc, her-2,TGF-α, and bcl-2.

As used herein, the term “under conditions such that growth of said cellis reduced” refers to conditions where an oligonucleotide of the presentinvention, when administered to a cell (e.g., a cancer) reduces the rateof growth of the cell by at least 10%, preferably at least 25%, evenmore preferably at least 50%, and still more preferably at least 90%relative to the rate of growth of the cell in the absence of theoligonucleotide.

The term “epitope” as used herein refers to that portion of an antigenthat makes contact with a particular antibody.

When a protein or fragment of a protein is used to immunize a hostanimal, numerous regions of the protein may induce the production ofantibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as “antigenic determinants”. An antigenic determinantmay compete with the intact antigen (i.e., the “immunogen” used toelicit the immune response) for binding to an antibody.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

As used herein, the terms “computer memory” and “computer memory device”refer to any storage media readable by a computer processor. Examples ofcomputer memory include, but are not limited to, RAM, ROM, computerchips, digital video disc (DVDs), compact discs (CDs), hard disk drives(HDD), and magnetic tape.

As used herein, the term “computer readable medium” refers to any deviceor system for storing and providing information (e.g., data andinstructions) to a computer processor. Examples of computer readablemedia include, but are not limited to, DVDs, CDs, hard disk drives,magnetic tape and servers for streaming media over networks.

As used herein, the terms “processor” and “central processing unit” or“CPU” are used interchangeably and refer to a device that is able toread a program from a computer memory (e.g., ROM or other computermemory) and perform a set of steps according to the program.

As used herein, the term “non-human animals” refers to all non-humananimals including, but are not limited to, vertebrates such as rodents,non-human primates, ovines, bovines, ruminants, lagomorphs, porcines,caprines, equines, canines, felines, aves, etc. and and non-vertebrateanimals such as drosophila and nematode. In some embodiments, “non-humananimals” further refers to prokaryotes and viruses such as bacterialpathogens, viral pathogens.

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule, including but not limited to, DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N-6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil,queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil,4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

As used herein, the term “heterologous gene” refers to a gene that isnot in its natural environment. For example, a heterologous geneincludes a gene from one species introduced into another species. Aheterologous gene also includes a gene native to an organism that hasbeen altered in some way (e.g., mutated, added in multiple copies,linked to non-native regulatory sequences, etc). Heterologous genes aredistinguished from endogenous genes in that the heterologous genesequences are typically joined to DNA sequences that are not foundnaturally associated with the gene sequences in the chromosome or areassociated with portions of the chromosome not found in nature (e.g.,genes expressed in loci where the gene is not normally expressed).

As used herein, the term “gene expression” refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of mRNA. Gene expression can beregulated at many stages in the process. “Up-regulation” or “activation”refers to regulation that increases the production of gene expressionproducts (i.e., RNA or protein), while “down-regulation” or “repression”refers to regulation that decrease production. Molecules (e.g.,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequencesthat are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

The term “wild-type” refers to a gene or gene product isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product that displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics (includingaltered nucleic acid sequences) when compared to the wild-type gene orgene product.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene” and “polynucleotide having a nucleotidesequence encoding a gene,” means a nucleic acid sequence comprising thecoding region of a gene or in other words the nucleic acid sequence thatencodes a gene product. The coding region may be present in a cDNA,genomic DNA or RNA form. When present in a DNA form, the oligonucleotideor polynucleotide may be single-stranded (i.e., the sense strand) ordouble-stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the expressionvectors of the present invention may contain endogenousenhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

As used herein, the term “oligonucleotide,” refers to a short length ofsingle-stranded polynucleotide chain. Oligonucleotides are typicallyless than 200 residues long (e.g., between 8 and 100), however, as usedherein, the term is also intended to encompass longer polynucleotidechains (e.g., as large as 5000 residues). Oligonucleotides are oftenreferred to by their length. For example a 24 residue oligonucleotide isreferred to as a “24-mer”. Oligonucleotides can form secondary andtertiary structures by self-hybridizing or by hybridizing to otherpolynucleotides. Such structures can include, but are not limited to,duplexes, hairpins, cruciforms, bends, and triplexes.

In some embodiments, oligonucleotides are “antigenes.” As used herein,the term “antigene” refers to an oligonucleotide that hybridizes to thepromoter region of a gene. In some embodiments, the hybridization of theantigene to the promoter inhibits expression of the gene.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, for the sequence“A-G-T,” is complementary to the sequence “T-C-A.” Complementarity maybe “partial,” in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules. Or, there may be “complete” or“total” complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods that depend upon binding between nucleicacids.

As used herein, the term “completely complementary,” for example whenused in reference to an oligonucleotide of the present invention refersto an oligonucleotide where all of the nucleotides are complementary toa target sequence (e.g., a gene).

As used herein, the term “partially complementary,” for example whenused in reference to an oligonucleotide of the present invention, refersto an oligonucleotide where at least one nucleotide is not complementaryto the target sequence. Preferred partially complementaryoligonucleotides are those that can still hybridize to the targetsequence under physiological conditions. The term “partiallycomplementary” refers to oligonucleotides that have regions of one ormore non-complementary nucleotides both internal to the oligonucleotideor at either end. Oligonucleotides with mismatches at the ends may stillhybridize to the target sequence.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is a nucleic acid molecule that at leastpartially inhibits a completely complementary nucleic acid molecule fromhybridizing to a target nucleic acid is “substantially homologous.” Theinhibition of hybridization of the completely complementary sequence tothe target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely homologous nucleic acid molecule to a target underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second target that issubstantially non-complementary (e.g., less than about 30% identity); inthe absence of non-specific binding the probe will not hybridize to thesecond non-complementary target.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described above.

A gene may produce multiple RNA species that are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene will contain regions of sequenceidentity or complete homology (representing the presence of the sameexon or portion of the same exon on both cDNAs) and regions of completenon-identity (for example, representing the presence of exon “A” on cDNA1 wherein cDNA 2 contains exon “B” instead). Because the two cDNAscontain regions of sequence identity they will both hybridize to a probederived from the entire gene or portions of the gene containingsequences found on both cDNAs; the two splice variants are thereforesubstantially homologous to such a probe and to each other.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low stringency as described above.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids. A single molecule that contains pairing of complementarynucleic acids within its structure is said to be “self-hybridized.”

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985]). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Under “low stringency conditions” anucleic acid sequence of interest will hybridize to its exactcomplement, sequences with single base mismatches, closely relatedsequences (e.g., sequences with 90% or greater homology), and sequenceshaving only partial homology (e.g., sequences with 50-90% homology).Under “medium stringency conditions,” a nucleic acid sequence ofinterest will hybridize only to its exact complement, sequences withsingle base mismatches, and closely relation sequences (e.g., 90% orgreater homology). Under “high stringency conditions,” a nucleic acidsequence of interest will hybridize only to its exact complement, and(depending on conditions such a temperature) sequences with single basemismatches. In other words, under conditions of high stringency thetemperature can be raised so as to exclude hybridization to sequenceswith single base mismatches.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5× Denhardt's reagent [50× Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5×SSPE, 0.1% SDS at 42° C. when a probe of about 500nucleotides in length is employed.

The present invention is not limited to the hybridization of probes ofabout 500 nucleotides in length. The present invention contemplates theuse of probes between approximately 8 nucleotides up to several thousand(e.g., at least 5000) nucleotides in length. One skilled in the relevantunderstands that stringency conditions may be altered for probes ofother sizes (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985] and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY[1989]).

The art knows well that numerous equivalent conditions may be employedto comprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)are considered and the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, the use of formamide in the hybridization solution, etc.)(see definition above for “stringency”).

As used herein, the term “physiological conditions” refers to specificstringency conditions that approximate or are conditions inside ananimal (e.g., a human). Exemplary physiological conditions for use invitro include, but are not limited to, 37° C., 95% air, 5% CO₂,commercial medium for culture of mammalian cells (e.g., DMEM mediaavailable from Gibco, Md.), 5-10% serum (e.g., calf serum or horseserum), additional buffers, and optionally hormone (e.g., insulin andepidermal growth factor).

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecomponent or contaminant with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is such present in a form orsetting that is different from that in which it is found in nature. Incontrast, non-isolated nucleic acids as nucleic acids such as DNA andRNA found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding a given protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the givenprotein where the nucleic acid is in a chromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid, oligonucleotide, or polynucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acid,oligonucleotide or polynucleotide is to be utilized to express aprotein, the oligonucleotide or polynucleotide will contain at a minimumthe sense or coding strand (i.e., the oligonucleotide or polynucleotidemay be single-stranded), but may contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide may bedouble-stranded).

As used herein, the term “purified” or “to purify” refers to the removalof components (e.g., contaminants) from a sample. For example,antibodies are purified by removal of contaminating non-immunoglobulinproteins; they are also purified by the removal of immunoglobulin thatdoes not bind to the target molecule. The removal of non-immunoglobulinproteins and/or the removal of immunoglobulins that do not bind to thetarget molecule results in an increase in the percent of target-reactiveimmunoglobulins in the sample. In another example, recombinantpolypeptides are expressed in bacterial host cells and the polypeptidesare purified by the removal of host cell proteins; the percent ofrecombinant polypeptides is thereby increased in the sample.

“Amino acid sequence” and terms such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

The term “native protein” as used herein to indicate that a protein doesnot contain amino acid residues encoded by vector sequences; that is,the native protein contains only those amino acids found in the proteinas it occurs in nature. A native protein may be produced by recombinantmeans or may be isolated from a naturally occurring source.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid.

The term “Southern blot,” refers to the analysis of DNA on agarose oracrylamide gels to fractionate the DNA according to size followed bytransfer of the DNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized DNA is then probedwith a labeled probe to detect DNA species complementary to the probeused. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists (J.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, NY, pp 9.31-9.58 [1989]).

The term “Northern blot,” as used herein refers to the analysis of RNAby electrophoresis of RNA on agarose gels to fractionate the RNAaccording to size followed by transfer of the RNA from the gel to asolid support, such as nitrocellulose or a nylon membrane. Theimmobilized RNA is then probed with a labeled probe to detect RNAspecies complementary to the probe used. Northern blots are a standardtool of molecular biologists (J. Sambrook, et al., supra, pp 7.39-7.52[1989]).

The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of radiolabeled antibodies.

As used herein, the term “cell culture” refers to any in vitro cultureof cells. Included within this term are continuous cell lines (e.g.,with an immortal phenotype), primary cell cultures, transformed celllines, finite cell lines (e.g., non-transformed cells), and any othercell population maintained in vitro.

As used, the term “eukaryote” refers to organisms distinguishable from“prokaryotes.” It is intended that the term encompass all organisms withcells that exhibit the usual characteristics of eukaryotes, such as thepresence of a true nucleus bounded by a nuclear membrane, within whichlie the chromosomes, the presence of membrane-bound organelles, andother characteristics commonly observed in eukaryotic organisms. Thus,the term includes, but is not limited to such organisms as fungi,protozoa, and animals (e.g., humans).

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments can consist of, but are not limitedto, test tubes and cell culture. The term “in vivo” refers to thenatural environment (e.g., an animal or a cell) and to processes orreaction that occur within a natural environment.

The terms “test compound” and “candidate compound” refer to any chemicalentity, pharmaceutical, drug, and the like that is a candidate for useto treat or prevent a disease, illness, sickness, or disorder of bodilyfunction (e.g., cancer). Test compounds comprise both known andpotential therapeutic compounds. A test compound can be determined to betherapeutic by screening using the screening methods of the presentinvention. In some embodiments of the present invention, test compoundsinclude antisense compounds.

As used herein, the term “known chemotherapeutic agents” refers tocompounds known to be useful in the treatment of disease (e.g., cancer).Exemplary chemotherapeutic agents affective against cancer include, butare not limited to, daunorubicin, dactinomycin, doxorubicin, bleomycin,mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide,6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU),floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine,vinblastine, etoposide, teniposide, cisplatin and diethylstilbestrol(DES).

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Environmental samplesinclude environmental material such as surface matter, soil, water,crystals and industrial samples. Such examples are not however to beconstrued as limiting the sample types applicable to the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for thetreatment of cancers. In particular, the present invention providesoligonucleotide-based therapeutics for the inhibition of oncogenesinvolved in a variety of cancers. The present invention is not limitedto the treatment of a particular cancer. Any cancer can be targeted,including, but not limited to, breast cancers. The present invention isalso not limited to the targeting of cancers or oncogenes. The methodsand compositions of the present invention are suitable for use with anygene that it is desirable to inhibit the expression of (e.g., fortherapeutic or research uses).

I. Oncogene Targets

In some embodiments, the present invention provides antigene inhibitorsof oncogenes. The present invention is not limited to the inhibition ofa particular oncogene. Indeed, the present invention encompassesantigene inhibitors to any number of oncogenes including, but notlimited to, those disclosed herein.

A. Ras

One gene which has captured the attention of many scientists is thehuman proto-oncogene, c-Ha-ras. The nucleic acid sequence of thepromoter region of c-H-ras is shown in FIG. 7. This gene acts as acentral dispatcher, relaying chemical signals into cells and controllingcell division. Ras gene alteration may cause the gene to stay in the“on” position. The ras oncogene is believed to underlie up to 30% ofcancer, including colon cancer, lung cancer, bladder and mammarycarcinoma (Bos, Cancer Res. 49:4682-4689 [1989]). The ras oncogene hastherefore become a target for therapeutic drugs.

There are several reports showing that oligonucleotides complementary tovarious sites of ras mRNA can inhibit synthesis of ras protein (p21),which decreases the cell proliferation rate in cell culture (U.S. Pat.No. 5,576,208; U.S. Pat. No. 5,582,986; Daska et al., Oncogene Res.5:267-275 [1990]; Brown et al., Oncogene Res. 4:243-252 [1989];Saison-Behmoaras et al., EMBO J. 10:1111-1116 [1991)]. Oligonucleotidescomplementary to the 5′ flanking region of the c-Ha-ras RNA transcripthave shown to inhibit tumor growth in nude mice for up to 14 days (Grayet al., Cancer Res. 53:577-580 [1993]). It was recently reported that anantisense oligonucleotide directed to a point mutation (G>C) in codon 12of the c-Ha-ras mRNA inhibited cell proliferation as well as tumorgrowth in nude mice when it was injected subcutaneously (U.S. Pat. No.5,576,208; U.S. Pat. No. 5,582,986; Schwab et al., Proc. Natl. Acad.Sci. USA 91:10460-10464 [1994]; each of which is herein incorporated byreference). Researchers have also reported that antisense drugs shrankovarian tumors in small clinical trials (Roush et al., Science276:1192-1194 [1997]).

B. Her-2

The HER-2 (also known as neu oncogene or erbB-2) oncogene encodes areceptor-like tyrosine kinase (RTK) that has been extensivelyinvestigated because of its role in several human carcinomas (Hynes andStern, Biochim. et Biophy. Acta 1198:165-184 [1994]; Dougall et al.,Oncogene 9:2109-2123 [1994]) and in mammalian development (Lee et al.,Nature 378:394-398 [1995]). The nucleic acid sequence of the promoterregion of Her-2 is shown in FIG. 3. The sequence of the HER-2 proteinwas determined from a cDNA that was cloned by homology to the epidermalgrowth factor receptor (EGFR) mRNA from placenta (Coussens et al.,Science 230:1132-1139 [1985]) and from a gastric carcinoma cell line(Yamamoto et al., Nature 319:230-234 [1986]). The HER-2 mRNA was shownto be about 4.5 kb (Coussens et al., Science 230:1132-1139 [1985];Yamamoto et al., Nature 319:230-234 [1986]) and encodes a transmembraneglycoprotein of 185 kDa in normal and malignant human tissues(p185HER-2) (Hynes and Steen, Biochim. et Biophys. Acta 1198:165-184[1994]; Dougall et al., Oncogene 9:2109-2123 [1994]). Overexpression ofHER-2 causes phenotypic transformation of cultured cells (DiFiore etal., Science 237:178-182 [1987]; Hudziak et al., Proc. Natl. Acad. Sci.USA 84:7159-7163 [1987]) and has been associated with aggressiveclinical progression of breast and ovarian cancer (Slamon et al.,Science 235:177-182 [1987]; Slamon et al., Science 244:707-712 [1989]).

HER-2 is one of the most frequently altered genes in cancer. It encodesa transmembrane receptor (also known as p185) with tyrosine kinaseactivity and is a member of the epidermal growth factor (EGF) family,and thus is related to the epidermal growth factor receptor (EGFR orHER-1). Aberrant HER-2 gene expression is present in a wide variety ofcancers and are most common in breast, ovarian and gastric cancers.HER-2 is overexpressed in 25-30% of all human breast and ovariancancers. Levels of HER-2 overexpression correlate well with clinicalstage of breast cancer, prognosis and metastatic potential.Overexpression of HER-2 is associated with lower survival rates,increased relapse rates and increased metastatic potential. Tan et al.,(Cancer Res., 57:1199 [1997]) have shown that overexpression of theHER-2 gene increases the metastatic potential of breast cancer cellswithout increasing their transformation ability.

Aberrant expression of HER-2 includes both increased expression ofnormal HER-2 and expression of mutant HER-2. Activation of the HER-2proto-oncogene can occur by any of three mechanisms—point mutation, geneamplification and overexpression. Gene amplification is the most commonmechanism. Unlike the other EGF family members for whom ligandactivation is necessary for promoting transformation, overexpression ofHER-2 alone is sufficient for transformation (Cohen, et al., J. Biol.Chem., 271:30897 [1996]).

Several therapeutic approaches have been used to reduce levels of theHER-2 gene product. The adenovirus type 5 gene product E1A has beenstudied as a potential therapeutic using a breast cancer model in nudemice. This gene product can repress HER-2/neu overexpression byrepressing HER-2/neu promoter activity, and suppress the tumorigenicpotential of HER-2/neu-overexpressing ovarian cancer cells. In micebearing HER-2/neu-overexpressing breast cancer xenografts, E1A deliveredeither by adenovirus or liposome significantly inhibited tumor growthand prolonged mouse survival compared with the controls (Chang et al.,Oncogene 14:561 [1997])

Clinical trials have been conducted to evaluate a bispecific antibodywhich targets the extracellular domains of both the HER-2/neu proteinproduct and Fc gamma RIII (CD16), the Fc gamma receptor expressed byhuman natural killer cells, neutrophils, and differentiated mononuclearphagocytes (Weiner et al., J. Hematotherapy, 4:471 [1995]).

Overexpression of HER-2 has also been found to be associated withincreased resistance to chemotherapy. Thus, patients with elevatedlevels of HER-2 respond poorly to many drugs. Methods used to inhibitHER-2 expression have been combined with commonly used chemotherapeuticagents (Ueno et al., Oncogone 15:953 [1997]). Combining the adenovirustype 5 gene product, E1A, with taxol showed a synergistic effect inhuman breast cancer cells. Zhang et al., (Oncogene, 12:571 [1996])demonstrated that emodin, a tyrosine-specific inhibitor, sensitizednon-small cell lung cancer (NSCLC) cells to a variety ofchemotherapeutic drugs, including cisplatin, doxorubicin and etoposide.A HER-2 antibody was found to increase the efficacy of tamoxifen inhuman breast cancer cells (Witters et al., Breast Cancer Res. andTreatment, 42:1 [1997]).

Oligonucleotides have also been used to study the function of HER-2. Atriplex-forming oligonucleotide targeted to the HER-2 promoter, 42 to 69nucleotides upstream of the mRNA transcription start site was found toinhibit HER-2 expression in vitro (Ebbinghaus et al., J. Clin. Invest.,92:2433 [1993]). Porumb et al. (Cancer Res., 56:515 [1996]) also used atriplex-forming oligonucleotide targeted to the same HER-2 promoterregion. Decreases in HER-2 mRNA and protein levels were seen in culturedcells. Juhl et al. (J. Biol. Chem., 272:29482 [1997]) used anti-HER-2ribozymes targeted to a central region of the HER-2 RNA just downstreamof the transmembrane region of the protein to demonstrate a reduction inHER-2 mRNA and protein levels in human ovarian cancer cells. A reductionin tumor growth in nude mice was also seen.

An antisense approach has been used as a potential therapeutic for HER-2overexpressing cancers. Pegues et al. (Cancer Lett., 117:73 [1997])cloned a 1.5 kb fragment of HER-2 in an antisense orientation into anexpression vector; transfecting of this construct into ovarian cancercells resulted in a reduction of anchorage-independent growth. Casaliniet al. (Int. J. Cancer 72:631 [1997]) used several human HER-2 antisensevector constructs, containing HER-2 fragments from 151 bp to 415 bp inlength, to demonstrate reduction in HER-2 protein levels andanchorage-independent growth in lung adenocarcinoma cells. Colomer etal. (Br. J. Cancer, 70:819 [1994]) showed that phosphodiester antisenseoligonucleotides targeted at or immediately downstream of, thetranslation initiation codon inhibited proliferation of human breastcancer cells by up to 60%. Wiechen et al. (Int. J. Cancer 63:604 [1995])demonstrated that an 18-nucleotide phosphorothioate oligonucleotidetargeted to the coding region, 33 nucleotides downstream of thetranslation initiation codon, of HER-2 reduced anchorage-independentgrowth of ovarian cancer cells. Bertram et al. (Biochem. Biophys. Res.Commun., 200:661 [1994]) used antisense phosphorothioateoligonucleotides targeted to the translation initiation region and asequence at the 3′ part of the translated region of the mRNA which hashigh homology to a tyrosine kinase consensus sequence, and demonstrateda 75% reduction in HER-2 protein levels in human breast cancer cells.Liu et al., (Antisense and Nucleic Acid Drug Develop., 6:9 [1996]) usedantisense phosphorothioate oligonucleotides targeted to the 5′ cap siteand coding region. The most effective oligonucleotide, targeted to the5′ cap site, reduced HER-2 protein expression by 90%. Cell proliferationwas also reduced by a comparable amount. Vaughn et al. (Nuc. Acids.Res., 24:4558 [1996]) used phosphorothioate, phosphorodithioate andchimeric antisense oligonucleotides targeted at or adjacent to (eitherside) the translation initiation region of HER-2. An alternatingdithioate/diester oligonucleotide targeted to the translation initiationregion worked slightly better than an all phosphorothioateoligonucleotide. Brysch et al. (Cancer Gene Ther., 1: 99 [1994]) usedchemically modified antisense oligonucleotides targeted to thetranslation initiation codon of HER-2 to reduce protein levels and causegrowth arrest of human breast cancer cell line.

C. C-Myc

The c-myc gene product is encoded by an immediate early response gene,the expression of which can be induced by various mitogens. The nucleicacid sequence of the promoter region of the c-myc gene is shown in FIG.9. C-myc expression is involved in the signal transduction pathwaysleading to cell division. Studies have demonstrated that proliferatingcells have higher levels of c-myc mRNA and c-myc protein than doquiescent cells. Antibodies directed against the human c-myc protein areknown to inhibit DNA synthesis in nuclei isolated from human cells.Conversely, constitutive expression of c-myc produced by gene transferinhibits induced differentiation of several cell lines. Constitutiveexpression of c-myc predisposes transgenic mice to the development oftumors.

Some studies have suggested that the c-myc gene product may play aproliferative role in SMCs. Balloon de-endothelialization and injury ofrat aortas is known to increase c-myc mRNA expression of vascular SMCprior to their subsequent proliferation and migration. Also, SMCs inculture proliferate when exposed to several mitogens, including PDGF,FGF, EGF, IGF-1 and to serum. Each of these mitogens has been found tobe capable of increasing the expression in other cell lines of eitherc-myc protein, c-myc mRNA, or both. Additionally, blood serum has beenfound to increase c-myc mRNA levels in SMCs.

Harel-Bellan et al. (J. Immun. 140; 2431-2435 (1988)) demonstrated thatantisense oligonucleotides complementary to c-myc mRNA effectivelyinhibited the translation thereof in human T cells. These T cells wereprevented from entering the S phase of cell division. c-mycproto-oncogene sequences are described in Marcu et al., Ann. Rev.Biochem., 61:809-860 [1992]; Watt et al., Nature, 303:725-728 [1983)];Battey et al., Cell, 34:779-787 (1983); and Epstein et al, NTISpublication PB93-100576

D. Bcl2

In many types of human tumors, including lymphomas and leukemias, thehuman bcl-2 gene is overexpressed, and may be associated withtumorigenicity (Tsujimoto et al., Science 228:1440-1443 [1985]). Thenucleic acid sequence of the promoter region of bcl-2 is shown inFIG. 1. High levels of expression of the human bcl-2 gene have beenfound in all lymphomas with t (14; 18) chromosomal translocationsincluding most follicular B cell lymphomas and many large cellnon-Hodgkin's lymphomas. High levels of expression of the bcl-2 genehave also been found in certain leukemias that do not have a t(14; 18)chromosomal translation, including most cases of chronic lymphocyticleukemia acute, many lymphocytic leukemias of the pre-B cell type,neuroblastomas, nasophryngeal carcinomas, and many adenocarcinomas ofthe prostate, breast, and colon. (Reed et al., Cancer Res. 51:6529[1991]; Yunis et al., New England J. Med. 320:1047; Campos et al., Blood81:3091-3096 [1993]; McDonnell et al., Cancer Res. 52:6940-6944 [1992);Lu et al., Int. J Cancer 53:29-35 [1993]; Bonner et al., Lab Invest.68:43A [1993]).

E. TGF-α

Transforming Growth Factor Alpha (TGF-α) is a polypeptide of 50 aminoacids. The nucleic acid sequence of the TGF-α promoter is shown in FIG.11. It was first isolated from a retrovirus-transformed mouse cell lineand subsequently was identified in human tumor cells, in early ratembryo cells and in cell cultures from the human pituitary gland. TGF-αis closely related to Epidermal Growth Factor (EGF), both structurallyand functionally, and both bind to the same receptor, i.e., EpidermalGrowth Factor Receptor (EGFR).

The sequence and three dimensional structure of both EGF and TGF-α havebeen determined (Campbell et al., Prog. Growth Factor Res. 1:13 [1989]).TGF-α is a 50 amino acid polypeptide having about 40% homology ofresidues with EGF. Both peptides are characterized by three well definedloops (denoted A, B and C) and have three intramolecular disulphidebonds.

Several growth factors, including TGF-α and EGF, are believed to exerttheir biological effects via interaction with the Epidermal GrowthFactor Receptor (EGF Receptor). The EGF Receptor is a Type 1 receptortyrosine kinase. The EGF Receptor and its ligands are of interest fortheir roles in normal physiological processes as well as inhyperproliferative and neoplastic diseases.

The in vivo precursor of TGF-α is a 160 amino acid residuemembrane-bound protein (pro-TGF-.alpha.) that is cleaved to yield asoluble compound (Massague, J. Biol. Chem., 265:21393-21396 [1990]).This cleavage removes an extracellular portion comprised of 50 aminoacids with a molecular weight of 6 Kd and is considered to be animportant regulatory event (Pandiella et al., Proc. Natl. Acad. Sci.USA, 88:1726-1730 [1990]) that can be stimulated by phorbol estersacting via protein kinase C (Pandiella et al., J. Biol. Chem.,266:5769-5773 [1991]).

Cultured human prostatic tumor lines contain elevated levels of TGF-αmRNA and proliferate in response to TGF-α (Wilding et al., The Prostate,15:1-12 [1989]). TGF-α appears to have both autocrine and paracrinefunction, stimulating physiologic activities such as cell division andangiogenesis. When induced in transgenic mice, TGF-α produced epithelialhyperplasia and focal dysplastic changes that resembled carcinoma insitu (Sandgren et al., Cell, 61:1121-1135 [1990]).

F. c-ki-RAS

The c-Ki-RAS (KRAS) oncogene is expressed ubiquitously. KRAS, with alength of more than 30 kb, is much larger than HRAS or NRAS. Thesequence of the promoter region of c-ki-ras is shown in FIG. 5. Althoughthe 3 ras genes, HRAS, KRAS, and NRAS, have different geneticstructures, all code for proteins of 189 amino acid residues,generically designated p21. These genes acquire malignant properties bysingle point mutations that affect the incorporation of the 12th or 61stamino acid residue of their respective p21. KRAS is involved inmalignancy much more often than is HRAS. In a study of 96 human tumorsor tumor cell lines in the NIH 3T3 transforming system, (Pulciani etal., Nature 300: 539 (1982) found a mutated HRAS locus only in T24bladder cancer cells, whereas transforming KRAS genes were identified in8 different carcinomas and sarcomas.

In a serous cystadenocarcinoma of the ovary, Feig et al. (Science 223:698 (1984)) showed the presence of an activated KRAS oncogene notactivated in normal cells of the same patient. The transforming geneproduct displayed an electrophoretic mobility in SDS-polyacrylamide gelsthat differed from the mobility of KRAS transforming proteins in othertumors. Thus, a previously undescribed mutation was responsible foractivation of KRAS in this ovarian carcinoma. To study the role ofoncogenes in lung cancer, Rodenhuis et al. (New Eng. J. Med. 317: 929(1987)) used an assay based on oligonucleotide hybridization followingan in vitro amplification step. Genomic DNA was examined from 39 tumorspecimens obtained at thoracotomy. The KRAS gene was found to beactivated by point mutations in codon 12 in 5 of 10 adenocarcinomas. Twoof these tumors were less than 2 cm in size and had not metastasized. NoHRAS, KRAS, or NRAS mutations were observed in 15 squamous cellcarcinomas, 10 large cell carcinomas, 1 carcinoid, 2 metastaticadenocarcinomas from primary tumors outside the lung, and 1 small cellcarcinoma. An approximately 20-fold amplification of the unmutated KRASgene was observed in a tumor that proved to be a solitary lungmetastasis of a rectal carcinoma. Yanez et al. (Oncogene 1:315 (1987))found mutations in codon 12 of the KRAS gene in 4 of 16 colon cancers, 2of 27 lung cancers, and 1 of 8 breast cancers; no mutations were foundat position 61. Of the 6 possible amino acid replacements in codon 12,all but one were represented in the 7 mutations identified.

G. Other Oncogene Targets

The present invention is not limited to the oncogenes described above.The methods of the present invention are suitable for use with anyoncogene with a known promoter region. Exemplary oncogenes included, butare not limited to, BCR/ABL, ABL1/BCR, ABL, BCL1, CD24, CDK4,EGFR/ERBB-1, HSTF1, INT1/WNT1, INT2, MDM2, MET, MYB, MYC, MYCN, MYCL1,RAF1, NRAS, REL, AKT2, APC, BCL2-ALPHA, BCL2-BETA, BCL3, BCR, BRCA1,BRCA2, CBL, CCND1, CDKN1A, CDKN1C, CDKN2A, CDKN2B, CRK, CRK-II,CSF1R/FMS, DBL, DDOST, DCC, DPC4/SMAD4, E-CAD, E2F1/RBAP, ELK1, ELK3,EPH, EPHA1, E2F1, EPHA3, ERG, ETS1, ETS2, FER, FGR, FLI1/ERGB2, FOS,FPS/FES, FRA1, FRA2, FYN, HCK, HEK, HER3/ERBB-2, ERBB-3, HER4/ERBB-4,HST2, INK4A, INK4B, JUN, JUNB, JUND, KIP2, KIT, KRAS2A, KRAS2B, LCK,LYN, MAS, MAX, MCC, MLH1, MOS, MSH2, MYBA, MYBB, NF1, NF2, P53, PDGFB,PIM1, PTC, RB1, RET, ROS1, SKI, SRC1, TAL1, TGFBR2, THRA1, THRB, TIAM1,TRK, VAV, VHL, WAF1, WNT2, WT1, YES1, ALK/NPM1, AMI1, AXL, FMS, GIP,GLI, GSP, HOX11, HST, IL3, INT2, KS3, K-SAM, LBC, LMO-1, LMO-2, L-MYC,LYL1, LYT-10, MDM-2, MLH1, MLL, MLM, N-MYC, OST, PAX-5, PMS-1, PMS-2,PRAD-1, RAF, RHOM-1, RHOM-2, SIS, TAL2, TAN1, TIAM1, TSC2, TRK, TSC1,STK11, PTCH, MEN1, MEN2, P57/KIP2, PTEN, HPC1, ATM, XPA/XPG, BCL6, DEK,AKAP13, CDH1, BLM, EWSR1/FLI1, FES, FGF3, FGF4, FGF6, FANCA, FLI1/ERGB2,FOSL1, FOSL2, GLI, HRAS1, HRX/MLLT1, HRX/MLLT2, KRAS2, MADH4, MAS1,MCF2, MLLT1/MLL, MLLT2/HRX, MTG8/RUNX1, MYCLK1, MYH11/CBFB, NFKB2,NOTCH1, NPM1/ALK, NRG/REL, NTRK1, PBX1/TCF3, PML/RARA, PRCA1, RUNX1,RUNX1/CBFA2T1, SET, TCF3/PBX1, TGFB1, TLX1, P53, WNT1, WNT2, WT1, αv-β3,PKCα, TNFα, Clusterin, Surviving, TGFβ, c-fos, c-SRC, and INT-1.

II. Non-Oncogene Targets

The present invention is not limited to the targeting of oncogenes. Themethods and compositions of the present invention find use in thetargeting of any gene that it is desirable to down regulate theexpression of. For example, in some embodiments, the genes to betargeted include, but are not limited to, an immunoglobulin or antibodygene, a clotting factor gene, a protease, a pituitary hormone, aprotease inhibitor, a growth factor, a somatomedian, a gonadotrophin, achemotactin, a chemokine, a plasma protein, a plasma protease inhibitor,an interleukin, an interferon, a cytokine, a transcription factor, or apathogen target (e.g., a viral gene, a bacterial gene, a microbial gene,a fungal gene).

Examples of specific genes include, but are not limited to, ADAMTS4,ADAMTS5, APOA1, APOE, APP, B2M, COX2, CRP, DDX25, DMC1, FKBP8, GH1, GHR,IAPP, IFNA1, IFNG, IL1, Il10, IL12, IL13, IL2, IL4, IL7, IL8, IPW,MAPK14, Mei1, MMP13, MYD88, NDN, PACE4, PRNP, PSEN1, PSEN2, RAD51,RAD51C, SAP, SNRPN, TLR4, TLR9, TTR, UBE3A, VLA-4, and PTP-1B, c-RAF,m-TOR, LDL, VLDL, ApoB-100, HDL, VEGF, rhPDGF-BB, NADs, ICAM-1, MUC1,2-dG, CTL, PSGL-1, E2F, NF-kB, HIF, and GCPRs.

In other embodiments and gene from a pathogen is targeted. Exemplarypathogens include, but are not limited to, Human Immunodeficiency virus,Hepatitis B virus, hepatitis C virus, hepatitis A virus, respiratorysyncytial virus, pathogens involved in severe acute respiratorysyndrome, west nile virus, and food borne pathogens (e.g., E. coli).

III. DNA Methylation

In some embodiments, the present invention provides oligonucleotidetherapeutics that are methylated at specific sites. The presentinvention is not limited to a particular mechanism. Indeed, anunderstanding of the mechanism is not necessary to practice the presentinvention. Nonetheless, it is contemplated that one mechanism for theregulation of gene activity is methylation of cytosine residues in DNA.5-methylcytosine (5-MeC) is the only naturally occurring modified basedetected in DNA (Ehrlick et al., Science 212:1350-1357 (1981)). Althoughnot all genes are regulated by methylation, hypomethylation at specificsites or in specific regions in a number of genes is correlated withactive transcription (Doerfler, Annu. Rev. Biochem. 52:93-124 [1984];Christman, Curr. Top. Microbiol. Immunol. 108:49-78 [1988]; Cedar, Cell34:5503-5513 [1988]). DNA methylation in vitro can prevent efficienttranscription of genes in a cell-free system or transient expression oftransfected genes. Methylation of C residues in some specificcis-regulatory regions can also block or enhance binding oftranscriptional factors or repressors (Doerfler, supra; Christman,supra; Cedar, Cell 34:5503-5513 (1988); Tate et al., Curr. Opin. Genet.Dev. 3:225-231 [1993]; Christman et al., Virus Strategies, eds.Doerfler, W. & Bohm, P. (VCH, Weinheim, N.Y.) pp. 319-333 [1993]).

Disruption of normal patterns of DNA methylation has been linked to thedevelopment of cancer (Christman et al., Proc. Natl. Acad. Sci. USA92:7347-7351 [1995]). The 5-MeC content of DNA from tumors and tumorderived cell lines is generally lower than normal tissues (Jones et al.,Adv. Cancer Res 40:1-30 [1983]). Hypomethylation of specific oncogenessuch as c-myc, c-Ki-ras and c-Ha-ras has been detected in a variety ofhuman and animal tumors (Nambu et al., Jpn. J. Cancer (Gann) 78:696-704[1987]; Feinberg et al., Biochem. Biophys. Res. Commun. 111:47-54[1983]; Cheah et al., JNC173:1057-1063 [1984]; Bhave et al.,Carcinogenesis (Lond) 9:343-348 [1988]. In one of the best studiedexamples of human tumor progression, it has been shown thathypomethylation of DNA is an early event in development of colon cancer(Goetz et al., Science 228:187-290 [1985]). Interference withmethylation in vivo can lead to tumor formation. Feeding of methylationinhibitors such as L-methionine or 5-azacytodine or severe deficiency of5-adenosine methionine through feeding of a diet depleted of lipotropeshas been reported to induce formation of liver tumors in rats (Wainfanet al., Cancer Res. 52:2071s-2077s [1992]). Studies show that extremelipotrope deficient diets can cause loss of methyl groups at specificsites in genes such as c-myc, ras and c-fos (Dizik et al.,Carcinogenesis 12:1307-1312 [1991]). Hypomethylation occurs despite thepresence of elevated levels of DNA MTase activity (Wainfan et al.,Cancer Res. 49:4094-4097 [1989]). Genes required for sustained activeproliferation become inactive as methylated during differentiation andtissue specific genes become hypomethylated and are active.Hypomethylation can then shift the balance between the two states. Insome embodiment, the present invention thus takes advantage of thisnaturally occurring phenomena, to provide compositions and methods forsite specific methylation of specific gene promoters, thereby preventingtranscription and hence translation of certain genes. In otherembodiments, the present invention provides methods and compositions forupregulating the expression of a gene of interest (e.g., a tumorsuppressor gene) by altering the gene's methylation patterns.

The present invention is not limited to the use of methylatedoligonucleotides. Indeed, the use of non-methylated oligonucleotides forthe inhibition of gene expression is specifically contemplated by thepresent invention. Experiments conducted during the course ofdevelopment of the present invention (See e.g., Example 8) demonstratedthat an unmethylated oligonucleotide targeted toward Bcl-2 inhibited thegrowth of lymphoma cells to a level that was comparable to that of amethylated oligonucleotide.

IV. Oligonucleotides

In some embodiments, the present invention provides antigeneoligonucleotides for inhibiting the expression of oncogenes. Exemplarydesign and production strategies for antigenes are described below. Thebelow description is not intended to limit the scope of antigenecompounds suitable for use in the present invention. One skilled in therelevant recognizes that additional antigenes are within the scope ofthe present invention.

A. Oligonucleotide Design

In some embodiments, oligonucleotides are designed based on preferreddesign criteria. Such oligonucleotides can then be tested for efficacyusing the methods disclosed herein. For example, in some embodiments,the oligonucleotides are methylated at least one, preferably at leasttwo, and even more preferably, all of the CpG islands. In otherembodiments, the oligonucleotides contain no methylation. The presentinvention is not limited to a particular mechanism. Indeed, anunderstanding of the mechanism is not necessary to practice the presentinvention. Nonetheless, it is contemplated that preferredoligonucleotides are those that have at least a 50% GC content and atleast 2 GC dinucleotides. It is preferred that oligonucleotides do notself hybridize. In some embodiments, oligonucleotides are designed withat least 1 A or T to minimize self hybridization. In some embodiments,commercially available computer programs are used to surveyoligonucleotides for the ability to self hybridize. Preferredoligonucleotides are at least 10, and preferably at least 15 nucleotidesand no more than 100 nucleotides in length. Particularly preferredoligonucleotides are 18-24 nucleotides in length. In some embodiments,oligonucleotides comprise the universal protein binding sequences CGCCCand CGCG or the complements thereof.

It is also preferred that the oligonucleotide hybridize to a promoterregion of a gene upstream from the TATA box of the promoter. It is alsopreferred that oligonucleotide compounds are not completely homologousto other regions of the human genome. The homology of theoligonucleotide compounds of the present invention to other regions ofthe genome can be determined using available search tools (e.g., BLAST,available at the Internet site of NCBI).

In some embodiments, oligonucleotides are designed to hybridize toregions of the promoter region of an oncogene known to be bound byproteins (e.g., transcription factors). Exemplary oligonucleotidecompounds of the present invention are shown in FIGS. 2, 4, 6, 8, 10,and 12. The present invention is not limited to the oligonucleotidesdescribed herein. Other suitable oligonucleotides may be identified(e.g., using the criteria described above). Exemplary oligonucleotidevariants of the disclosed oligonucleotides are shown in FIGS. 25-30.Candidate oligonucleotides may be tested for efficacy using any suitablemethod, including, but not limited to, those described in theillustrative examples below. Using the in vitro assay described inExamples 1 and 2 below, candidate oligonucleotides can be evaluated fortheir ability to prevent cell proliferation as a variety ofconcentrations. Particularly preferred oligonucleotides are those thatinhibit gene expression of cell proliferation as a low concentration(e.g., less that 20 μM, and preferably, less than 10 μM in the in vitroassays disclosed herein).

B. Preferred Oligonucleotide Zones

In some embodiments, regions within the promoter region of an oncogeneare further defined as preferred regions for hybridization ofoligonucleotides. In some embodiments, these preferred regions arereferred to as “hot zones.”

In some preferred embodiments, hot zones are defined based onoligonucleotide compounds that are demonstrated to be effective (seeabove section on oligonucleotides) and those that are contemplated to beeffective based on the preferred criteria for oligonucleotides describedabove. Preferred hot zones encompass 10 bp upstream and downstream ofeach compound included in each hot zone and have at least 1 CG or morewithin an increment of 40 bp further upstream or downstream of eachcompound. In preferred embodiments, hot zones encompass a maximum of 100bp upstream and downstream of each oligonucleotide compound included inthe hot zone. In additional embodiments, hot zones are defined atbeginning regions of each promoter. These hot zones are defined eitherbased on effective sequence(s) or contemplated sequences and have apreferred maximum length of 200 bp. Based on the above describedcriteria, exemplary hot zones were designed. These hot zones are shownin Table 1. Numbering is based on the sequences described in the Figuresof the present invention. TABLE 1 Exemplary Hot Zones Gene Hot ZonesBcl-2  1-40  161-350 401-590 1002-1260 c-erbB-2 205-344 382-435 c-K-ras 1-289 432-658 c-Ha-ras  21-220 233-860 1411-1530 1631-1722 c-myc  3-124165-629 TGF-α  1-90  175-219 261-367 431-930  964-1237

C. Preparation and Formulation of Oligonucleotides

Any of the known methods of oligonucleotide synthesis can be used toprepare the modified oligonucleotides of the present invention. In someembodiments utilizing methylated oligonucleotides the nucleotide, dC isreplaced by 5-methyl-dC where appropriate, as taught by the presentinvention. The modified or unmodified oligonucleotides of the presentinvention are most conveniently prepared by using any of thecommercially available automated nucleic acid synthesizers. They canalso be obtained from commercial sources that synthesize customoligonucleotides pursuant to customer specifications.

While oligonucleotides are a preferred form of compound, the presentinvention comprehends other oligomeric oligonucleotide compounds,including but not limited to oligonucleotide mimetics such as aredescribed below. The oligonucleotide compounds in accordance with thisinvention preferably comprise from about 18 to about 30 nucleobases(i.e., from about 18 to about 30 linked bases), although both longer andshorter sequences may find use with the present invention.

Specific examples of preferred compounds useful with the presentinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, modifiedoligonucleotides that do not have a phosphorus atom in theirinternucleoside backbone can also be considered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage (i.e., the backbone) of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science 254:1497 (1991).

In some embodiments, oligonucleotides of the invention areoligonucleotides with phosphorothioate backbones and oligonucleosideswith heteroatom backbones, and in particular —CH₂, —NH—O—CH₂—,—CH₂—N(CH₃)—O—CH₂—[known as a methylene (methylimino) or MMI backbone],—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂—, and—O—N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone isrepresented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No.5,489,677, and the amide backbones of the above referenced U.S. Pat. No.5,602,240. Also preferred are oligonucleotides having morpholinobackbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta 78:486[1995]) i.e., an alkoxyalkoxy group. A further preferred modificationincludes 2′-dimethylaminooxyethoxy (i.e., a O(CH₂)₂ON(CH₃)₂ group), alsoknown as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (also known in theart as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other preferred modifications include 2′-methoxy(2′-O—CH₃),2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808. Certainof these nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Another modification of the oligonucleotides of the present inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates that enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety, cholic acid,a thioether, (e.g., hexyl-S-tritylthiol), a thiocholesterol, analiphatic chain, (e.g., dodecandiol or undecyl residues), aphospholipid, (e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or apolyethylene glycol chain or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

One skilled in the relevant art knows well how to generateoligonucleotides containing the above-described modifications. Thepresent invention is not limited to the antisense oligonucleotidesdescribed above. Any suitable modification or substitution may beutilized.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes pharmaceutical compositions and formulations that include theantisense compounds of the present invention as described below.

D. Cocktails

In some embodiments, the present invention provides cocktails comprisingtwo or more oligonucleotides directed towards promoter regions of genes(e.g., oncogenes). In some embodiments, the two oligonucleotideshybridize to different regions of the promoter of the same gene. Inother embodiments, the two or more oligonucleotides hybridize topromoters of two different genes. The present invention is not limitedto a particular mechanism. Indeed, an understanding of the mechanism isnot necessary to practice the present invention. Nonetheless, it iscontemplated that the combination of two or more compounds of thepresent invention provides an inhibition of cancer cell growth that isgreater than the additive inhibition of each of the compoundsadministered separately.

V. Research Uses

The present invention is not limited to therapeutic applications. Forexample, in some embodiments, the present invention providescompositions and methods for the use of oligonucleotides as a researchtool.

A. Kits

For example, in some embodiments, the present invention provides kitscomprising oligonucleotides specific for inhibition of a gene ofinterest, and optionally cell lines (e.g., cancer cells lines) known toexpress the gene. Such kits find use, for example, in the identificationof metabolic pathways or the involvement of genes in disease (e.g.,cancer), as well as in diagnostic applications. In some embodiments, thekits further comprise buffer and other necessary reagents, as well asinstructions for using the kits.

B. Target Validation

In some embodiments, the present invention provides methods andcompositions for use in the validation of gene targets (e.g., genessuspected of being involved in disease). For example, in someembodiments, the expression of genes identified in broad screeningapplications (e.g., gene expression arrays) as being involved in diseaseis downregulated using the methods and compositions of the presentinvention. The methods and compositions of the present invention aresuitable for use in vitro and in vivo (e.g., in a non-human animal) forthe purpose of target validation. In other embodiments, the compounds ofthe present invention find use in transplantation research (e.g., HLAinhibition).

C. Drug Screening

In other embodiments, the methods and compositions of the presentinvention are used in drug screening applications. For example, in someembodiments, oligonucleotides of the present invention are administeredto a cell (e.g., in culture or in a non-human animal) in order toinhibit the expression of a gene of interest. In some embodiments, theinhibition of the gene of interest mimics a physiological or diseasecondition. In other embodiments, an oncogene is inhibited. Testcompounds (e.g., small molecule drugs or oligonucleotide mimetics) arethen administered to the test cell and the effect of the test compoundsis assayed.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone, which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckennann et al., J.Med. Chem. 37: 2678-85 [1994]); spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are preferred for use withpeptide libraries, while the other four approaches are applicable topeptide, non-peptide oligomer or small molecule libraries of compounds(Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci.U.S.A. 90:6909 [1993]; Erb et al., Proc. Nad. Acad. Sci. USA 91:11422[1994]; Zuckermann et al., J. Med. Chem. 37:2678 [1994]; Cho et al.,Science 261:1303 [1993]; Carrell et al., Angew. Chem. Int. Ed. Engl.33.2059 [1994]; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061[1994]; and Gallop et al., J. Med. Chem. 37:1233 [1994].

Libraries of compounds may be presented in solution (e.g., Houghten,Biotechniques 13:412-421 [1992]), or on beads (Lam, Nature 354:82-84[1991]), chips (Fodor, Nature 364:555-556 [1993]), bacteria or spores(U.S. Pat. No. 5,223,409; herein incorporated by reference), plasmids(Cull et al., Proc. Nad. Acad. Sci. USA 89:18651869 [1992]) or on phage(Scott and Smith, Science 249:386-390 [1990]; Devlin Science 249:404-406[1990]; Cwirla et al., Proc. NatI. Acad. Sci. 87:6378-6382 [1990];Felici, J. Mol. Biol. 222:301 [1991]).

VI. Compositions and Delivery

In some embodiments, the oligonucleotide compounds of the presentinvention are formulated as pharmaceutical compositions for delivery toa subject as a pharmaceutical. The novel antigen compounds of thepresent invention find use in the treatment of a variety of diseasestates and conditions in which it is desirable to inhibit the expressionof a gene or the growth of a cell. In some preferred embodiments, thecompounds are used to treat disease states resulting from uncontrolledcell growth, for example including, but not limited to, cancer. Thepresent invention is not limited to the treatment of a particularcancer. The oligonucleotide compounds of the present invention aresuitable for the treatment of a variety of cancers including, but notlimited to, breast, colon, lung, stomach, pancreatic, bladder, leukemia,and lymphoma. The below discussion provides exemplary, non-limitingexamples of formulations and dosages.

A. Pharmaceutical Compositions

The present invention further provides pharmaceutical compositions(e.g., comprising the oligonucleotide compounds described above). Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances that increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product.

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (WO 97/30731), also enhancethe cellular uptake of oligonucleotides.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Prefered bileacids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate. Preferedfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also prefered are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly prefered combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyomithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG).

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more oligonucleotide compounds and (b) one or moreother chemotherapeutic agents that function by a non-oligonucleotidemechanism. Examples of such chemotherapeutic agents include, but are notlimited to, anticancer drugs such as daunorubicin, dactinomycin,doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil,melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine(CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. Other non-oligonucleotide chemotherapeutic agents are alsowithin the scope of this invention. Two or more combined compounds maybe used together or sequentially.

B. Delivery

The oligonucleotide compounds of the present invention may be deliveredusing any suitable method. In some embodiments, naked DNA isadministered. In other embodiments, lipofection is utilized for thedelivery of nucleic acids to a subject. In still further embodiments,oligonucleotides are modified with phosphothiolates for delivery (Seee.g., U.S. Pat. No. 6,169,177, herein incorporated by reference).

In some embodiments, nucleic acids for delivery are compacted to aid intheir uptake (See e.g., U.S. Pat. Nos. 6,008,366, 6,383,811 hereinincorporated by reference). In some embodiment, compacted nucleic acidsare targeted to a particular cell type (e.g., cancer cell) via a targetcell binding moiety (See e.g., U.S. Pat. Nos. 5,844,107, 6,077,835, eachof which is herein incorporated by reference).

In some embodiments, oligonucleotides are conjugated to other compoundsto aid in their delivery. For example, in some embodiments, nucleicacids are conjugated to polyethylene glycol to aid in delivery (Seee.g., U.S. Pat. Nos. 6,177,274, 6,287,591, 6,447,752, 6,447,753, and6,440,743, each of which is herein incorporated by reference). In yetother embodiments, oligonucleotides are conjugated to protected graftcopolymers, which are chargeable” drug nano-carriers (PharmaIn). Instill further embodiments, the transport of oligonucleotides into cellsis facilitated by conjugation to vitamins (Endocyte, Inc, WestLafayette, Ind.; See e.g., U.S. Pat. Nos. 5,108,921, 5,416,016,5,635,382, 6,291,673 and WO 02/085908; each of which is hereinincorporated by reference). In other embodiments, oligonucleotides areconjugated to nanoparticles (e.g., NanoMed Pharmaceuticals; Kalamazoo,Mich.).

In other embodiments, oligonucleotides are enclosed in lipids (e.g.,liposomes or micelles) to aid in delivery (See e.g., U.S. Pat. Nos.6,458,382, 6,429,200; each of which is herein incorporated byreference). In still further embodiments, oligonucleotides are complexedwith additional polymers to aid in delivery (See e.g., U.S. Pat. Nos.6,379,966, 6,339,067, 5,744,335; each of which is herein incorporated byreference and Intradigm Corp., Rockville, Md.).

In still further embodiments, the controlled high pressure deliverysystem developed by Mirus (Madison, Wis.) is utilized for delivery ofoligonucleotides.

C. Dosages

Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient. Theadministering physician can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and the deliverymeans, and can generally be estimated based on EC₅₀s found to beeffective in in vitro and in vivo animal models or based on the examplesdescribed herein. In general, dosage is from 0.01 μg to 100 g per kg ofbody weight, and may be given once or more daily, weekly, monthly oryearly. In some embodiments, dosage is continuous (e.g., intravenously)for a period of from several hours to several days or weeks. In someembodiments, treatment is given continuously for a defined periodfollowed by a treatment free period. In some embodiments, the pattern ofcontinuous dosing followed by a treatment free period is repeatedseveral times (e.g., until the disease state is diminished).

The treating physician can estimate repetition rates for dosing based onmeasured residence times and concentrations of the drug in bodily fluidsor tissues. Following successful treatment, it may be desirable to havethe subject undergo maintenance therapy to prevent the recurrence of thedisease state, wherein the oligonucleotide is administered inmaintenance doses, ranging from 0.01 μg to 100 g, preferably from 1 mgto 50 mg, and even more preferably from 6 mg to 30 mg per kg of bodyweight, once or more daily, to once every 20 years.

VII. Customized Patient Care

In some embodiments, the present invention provides customized patientcare. The compositions of the present invention are targeted to specificgenes unique to a patient's diseae (e.g., cancer). For example, in someembodiments, a sample of the patient's cancer or other affected tissue(e.g., a biopsy) is first obtained. The biopsy is analyzed for thepresence of expression of a particular gene (e.g., oncogene). In somepreferred embodiments, the level of expression of an gene in a patientis analyzed. Expression may be detected by monitoring for the presenceof RNA or DNA corresponding to a particular oncogene. Any suitabledetection method may be utilized, including, but not limited to, thosedisclosed below.

Following the characterization of the gene expression pattern of apatient's gene of interest, a customized therapy is generated for eachpatient. In preferred embodiments, oligonucleotide compounds specificfor genes that are aberrantly expressed in the patient (e.g., in atumor) are combined in a treatment cocktail. In some embodiments, thetreatment cocktail further includes additional chemotherapeutic agents(e.g., those described above). The cocktail is then administered to thepatient as described above.

In some embodiments, the analysis of cancer samples and the selection ofoligonucleotides for a treatment compound is automated. For example, insome embodiments, a software program that analyses the expression levelsof a series of oncogenes to arrive at the optimum selection andconcentration of oligonucleotides is utilized. In some embodiments, theanalysis is performed by the clinical laboratory analyzing the patientsample and is transmitted to a second provider for formulation of thetreatment cocktail. In some embodiments, the information is transmittedover the Internet, thus allowing for the shortest possible time inbetween diagnosis and the beginning of treatment.

A. Detection of RNA

In some embodiments, detection of oncogenes (e.g., including but notlimited to, those disclosed herein) is detected by measuring theexpression of corresponding mRNA in a tissue sample (e.g., cancertissue). In other embodiments, expression of mRNA is measured in bodilyfluids, including, but not limited to, blood, serum, mucus, and urine.In some preferred embodiments, the level of mRNA expression in measuredquantitatively. RNA expression may be measured by any suitable method,including but not limited to, those disclosed below.

In some embodiments, RNA is detected by Northern blot analysis. Northernblot analysis involves the separation of RNA and hybridization of acomplementary labeled probe. In other embodiments, RNA expression isdetected by enzymatic cleavage of specific structures (INVADER assay,Third Wave Technologies; See e.g., U.S. Pat. Nos. 5,846,717, 6,090,543;6,001,567; 5,985,557; and 5,994,069; each of which is hereinincorporated by reference). The INVADER assay detects specific nucleicacid (e.g., RNA) sequences by using structure-specific enzymes to cleavea complex formed by the hybridization of overlapping oligonucleotideprobes.

In still further embodiments, RNA (or corresponding cDNA) is detected byhybridization to a oligonucleotide probe). A variety of hybridizationassays using a variety of technologies for hybridization and detectionare available. For example, in some embodiments, TaqMan assay (PEBiosystems, Foster City, Calif.; See e.g., U.S. Pat. Nos. 5,962,233 and5,538,848, each of which is herein incorporated by reference) isutilized. The assay is performed during a PCR reaction. The TaqMan assayexploits the 5′-3′ exonuclease activity of the AMPLITAQ GOLD DNApolymerase. A probe consisting of an oligonucleotide with a 5′-reporterdye (e.g., a fluorescent dye) and a 3′-quencher dye is included in thePCR reaction. During PCR, if the probe is bound to its target, the 5′-3′nucleolytic activity of the AMPLITAQ GOLD polymerase cleaves the probebetween the reporter and the quencher dye. The separation of thereporter dye from the quencher dye results in an increase offluorescence. The signal accumulates with each cycle of PCR and can bemonitored with a fluorimeter.

In yet other embodiments, reverse-transcriptase PCR (RT-PCR) is used todetect the expression of RNA. In RT-PCR, RNA is enzymatically convertedto complementary DNA or “cDNA” using a reverse transcriptase enzyme. ThecDNA is then used as a template for a PCR reaction. PCR products can bedetected by any suitable method, including but not limited to, gelelectrophoresis and staining with a DNA specific stain or hybridizationto a labeled probe. In some embodiments, the quantitative reversetranscriptase PCR with standardized mixtures of competitive templatesmethod described in U.S. Pat. Nos. 5,639,606, 5,643,765, and 5,876,978(each of which is herein incorporated by reference) is utilized.

B. Detection of Protein

In other embodiments, gene expression of oncogenes is detected bymeasuring the expression of the corresponding protein or polypeptide. Insome embodiments, protein expression is detected in a tissue sample. Inother embodiments, protein expression is detected in bodily fluids. Insome embodiments, the level of protein expression is quantitated.Protein expression may be detected by any suitable method. In someembodiments, proteins are detected by their binding to an antibodyraised against the protein. The generation of antibodies is well knownto those skilled in the art.

Antibody binding is detected by techniques known in the art (e.g.,radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (e.g., usingcolloidal gold, enzyme or radioisotope labels, for example), Westernblots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays, etc.), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many methods are known in the art for detecting binding in animmunoassay and are within the scope of the present invention.

In some embodiments, an automated detection assay is utilized. Methodsfor the automation of immunoassays include those described in U.S. Pat.Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which isherein incorporated by reference. In some embodiments, the analysis andpresentation of results is also automated. For example, in someembodiments, software that generates an expression profile based on thepresence or absence of a series of proteins corresponding to oncogenesis utilized.

In other embodiments, the immunoassay described in U.S. Pat. Nos.5,599,677 and 5,672,480; each of which is herein incorporated byreference.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: N (normal); M (molar); mM (millimolar); μM(micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); l or L (liters); ml (milliliters); μl(microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm(nanometers); and ° C. (degrees Centigrade).

Example 1

Materials and Methods

This Example describes experimental methods utilized in the belowexamples.

A. Cell Lines

Cell lines used in experiments of the present invention are describedbelow.

MDA-MB-231

-   Tissue: adenocarcinoma; mammary gland; breast; pleural effusion-   Tumorigenic: forms adenocarcinoma grade III-   Receptors expressed: Epidermal Growth Factor (EGF) and Transforming    growth factor (TGF-alpha)-   Oncogene: wnt3+ and wnt7h+    References:-   Siciliano M J, Barker P E, Cailleau R. Mutually exclusive genetic    signatures of human breast tumor cell lines with a common    chromosomal marker.-    Cancer Res. 1979 March; 39(3):919-22.-   Calleau R, Olive M, Cruciger Q V. Long-term human breast carcinoma    cell lines of metastatic origin: preliminary characterization.-    In vitro. 1978 November; 14(11):911-5.-   Cruciger Q V, Pathak S, Calleau R. Human breast carcinomas: marker    chromosomes involving 1q in seven cases.-    Cytogenet Cell Genet. 1976; 17(4):231-5.-   Satya-Prakash K L, Pathak S, Hsu T C, Olive M, Cailleau R.-    Cytogenetic analysis on eight human breast tumor cell lines: high    frequencies of 1q, 11q and HeLa-like marker chromosomes. Cancer    Genet Cytogenet 1981 January; 3(1):61-73    MCF7-   Tissue: adenocarcinoma, mammary gland, breast-   Metastatic site: pleural effusion-   Receptors: estrogen receptor+-   Oncogenes: wnt7h+    This cell line is also known to moderately express c-erbB-2 and    overexpress c-myc oncogene-   Cellular product: Insulin like growth factor binding protein (IGFBP)    References:-   Soule H D et al. Ahuman cell line from a pleural effusion derived    from a breast carcinoma. J. Natl. Cancer Inst. 51: 1409-1416, 1973-   Landers J E et al. Translational enhancement of mdm2 oncogene    expression in human tumor cells containing a stabilized wild-type    p53 protein. Cancer Res. 57: 3562-3568, 1997-   Bacus S S et al. Differentiation of cultured human cancer cells    (AU-565 and MCF7) associated with loss of cell surface HER-2/neu    oligonucleotide. Mol. Carcinog. 3: 350-362, 1990    MCF10CA1

MCF10 cells are derived from benign breast tissue from a woman withfibrocystic disease. MCF10 lines consists of several lines, one isMCF10A, an immortalized normal human breast cell line. MCF10A wastransformed with T24 Ha-ras to make MCF10AneoT cells. MCF10AT withneoplastic progression potential was derived from xenograft passagedMCF10-AneoT. MCF10AT generates carcinoma in about 25% of xenografts.Fully malignant MCF10CA1 lines were derived from several xenograftpassages of MCF10AT. MCF10CA1a forms tumors 100% of the time and itmetastasizes. A kariotype of MCF10CA1a shows an extra copy ofchromosome 1. It metastasizes into the lung 36 days after IV injectionof the cells.

References:

-   Santner S J et al. Malignant MCF10CA1 cell lines derived from    premalignant human breast epithelial MCF1 OAT cells. Breast Cancer    Research and treatment 65: 101-110, 2001.    MYC-MT-1

A female MMTV-C-MYC transgenic mouse developed a mammary tumor. Thetumor was isolated and a small fresh tissue is put into culture with amedium conditioned by Dr. Jushoa Liao at Karmanos Cancer Institute. Thistumor cell line was established after 10 passages.

NMuMG

-   Tissue: Mouse normal mammary gland, epithelial-   Strain: NAMRU, female-   Tumorigenic: produce benign tumor in mice    References:-   Owens R B. Glandular epithelial cells from mice: a method for    selective cultivation. J. Natl. Cancer Inst. 52: 1375-1378, 1974-   Owens R B et al. Epithelial cell cultures from normal glandular    tissue of mice. J. Natl. Cancer Inst. 53: 261-269, 1974-   Yingling J M et al. Mammalian dwarfins are phosphorylated in    response to transforming growth factor beta and are implicated in    control of cell growth. Proc. Natl. Acad. Sci. USA 93: 8940-8944,    1996    BxPC-3-   Tissue: adenocarcinoma, pancreas    Cellular product: mucin, pancreatic cancer specific antigen; CEA,    carcinoma embryonic antigen.-   Source: 61 year old female-   Tumorigenic: yes-   Oncogenes: c-Ki-ras    References:-   Tan M H et al. Characterization of a new primary human pancreatic    tumor line. Cancer Invest. 4: 15-23, 1986-   Loor R et al. Use of pancreas-specific antigen in immunodiagnosis of    pancreatic cancer. Clin. Lab. Med. 2: 567-578, 1982-   Lan M S et al. Polypeptide core of a human pancreatic tumor mucin    antigen. Cancer Res. 50: 2997-3001, 1990-   Chambers J A and Harris A. Expression of the cystic fibrosis gene    and the major pancreatic mucin gene, MUC1, in human ductal    epithelial cells. J. Cell Sci. 105: 417-422, 1993    T-47D-   Tissue: ductal carcinoma, mammary gland, breast, duct-   Metastatic site: pleural effusion-   Source: pleural effusion of a 54 years old female with infiltrating    ductal carcinoma of the breast-   Receptor expression: estrogen, androgen, calcitonin, progesteron,    glucocorticoid and prolactin positive.-   Oncogenes: wnt3+ and wnt7h+-    This cell line is also know to overexpress c-erbB-2    References:-   Keydar I et al., Establishment and characterization of a cell line    of human breast carcinoma origin. Eur. J. Cancer 15: 659-670, 1979-   Judge S M and Chatterton R T Jr. Progesterone-specific stimulation    of triglyceride biosynthesis in a breast cancer cell line (T-47D).    Cancer Res. 43: 4407-4412, 1983-   Lamp S J et al. Calcitonin induction of a persistent activated state    of adenylate cyclase in human breast cancer cells (T-47D). J. Biol.    Chem. 256: 12269-12274, 1981-   Sher E et al. Whole-cell uptake and nuclear localization of    1,25-dihydroxy-cholecalciferol by breast cancer cells (T-47D) in    culture. Biochem. J. 200: 315-320, 1981-   Freake H C et al. 1,25-Dihydroxyvitamin D3 specifically binds to a    human breast cancer cell line (T-47D) and stimulates growth.    Biochem. Biophys. Res. Commun. 101: 1131-1138, 1981-   Faust J B and Meeker T C. Amplification and expression of the bcl-1    gene in human solid tumor cell lines. Cancer Res. 52: 2460-2463,    1992 RF33514:-   Huguet E L et al. Differential expression of human Wnt genes 2, 3,    4, and 7B in human breast cell lines and normal and disease states    of human breast tissue. Cancer Res. 54: 2615-2621, 1994    BT-474-   Tissue: ductal carcinoma, mammary gland, breast-   Source: 60 year old female-   Oncogene: c-erbB-2    References:-   Lasfargues E Y et al. Isolation of two human tumor epithelial cell    lines from solid breast carcinomas. J. Natl. Cancer Inst. 61:    967-978, 1978-   Lasfargues E Y et al. A human breast tumor cell line (BT-474) that    supports mouse mammary tumor virus replication. In vitro 15:    723-729, 1979-   Littlewood-Evans A J et al. The osteoclast-associated protease    cathepsin K is expressed in human breast carcinoma. Cancer Res. 57:    5386-5390, 1997    WSU-FSCCL    Human B-cell line established in 1993-   Source: from peripheral blood of a male patient with low grade    follicular small cleaved cell lymphoma in leukemic phase.-   Oncogenes: exhibiting chromosomal translocation for both c-myc and    bcl-2    References:-   Mohammad R M, Mohamed A N, Smith M R, Jawadi N S, AL-Khatib A. A    unique EBV-Negative Low Grade Lymphoma Line (WSU-FSCCL) Exhibiting    both t(14;18) and t(8;11). Cancer Genet Cytogenet 70:62-67, 1993    B. Cell Culture

Human breast cancer cells, MCF7, MCF10CAla, MDA-MB 231, MDA-MB 435.eB,and human normal breast cells, MCF10A were all obtained from KarmanosCancer Institute. All cells were cultured in DMEM/F12 media (Gibco, Md.)supplemented with 10 mM HEPES, 29 mM sodium bicarbonate, penicillin (100units/ml) and streptomycin (100 μg/ml). In addition, 10% calf serum, 10μg/ml insulin (Sigma Chemical, St Louis, Mo.), and 0.5 nM estradiol wasused in MCF7 media. 5% horse serum and insulin (10 μg/ml) was used forMCF10Cala, and 10% fetal calf serum was used for MDA-MB 231 and 435lines. MCF 1.0A culture was supplemented with 5% horse serum, insulin.(10 μg/ml), 100 ng/ml cholera enterotoxin (Calbiochem, Calif.), 0.5μg/ml hydrocortisone (Sigma Chemical) and 20 ng/ml epidermal growthfactor (Sigma Chemical). All flasks and plates were incubated in ahumidified atmosphere of 95% air and 5% CO₂ at 37° C.

MYC-MT-1 cells were also cultured in DMEM/F12 media containing 10 ng/mlEGF (epithelial growth factor), 1 nM estradiol, 10 μg/ml insulin and 10%FBS (fetal bovine serum). BxPC-3 pancreatic carcinoma cell line andBT-474, breast tumor cell line were cultured in RPMI 1640 with 10% FBS.Breast tumor cell line, T-47D was cultured in the same media as BT-474with the addition of 2.5 μg/ml insulin. NMuMG (normal mouse mammarygland cells) cell line was grown in DMEM media with 4.5 g/l glucose, 10μg/ml insulin and 10% FBS.

All the above cells were seeded at 2500 to 5,000 cells/well in 96 wellplates. The cells were treated with oligonucleotide compounds in freshmedia (100 μl total volume) 24 hours after seeding. The media wasreplaced with fresh media without oligonucleotides 24 hours aftertreatment and every 48 hours for 6 to 7 days or until the control cellswere 80 to 100% confluent. The inhibitory effect of oligonucleotide wasevaluated using an MTT staining technique.

Human follicular lymphoma cell line, WSU-FSCCL was used to evaluate theeffect of antic-myc oligonucleotides as well as anti-Bcl-2oligonucleotides. FSCCL cells grow as a single cell suspension in tissueculture. The culture was maintained in RPMI 1640 supplemented with 10%fetal bovine serum, 1% L-glutamine, 100 units/ml penicillin and 1.00μg/ml streptomycin. FSCCL cells were treated in 24 well plates (2×10⁵cells/well/ml) with oligonucleotide compounds and incubated in ahumidified atmosphere of 95% air and 5% CO₂ at 37° C. The cells werecounted every 24 hours using a hemocytometer.

C. Oligonucleotide Preparation

All oligonucleotides were synthesized, gel purified anal lyophilized byBIOSYNTHESIS (Lewisville, Tex.) or Qiagen (Valencia, Calif.). Methylatedoligonucleotides were methylated at all CpG sites. MethylatedOligonucleotides were dissolved in pure sterile water (Gibco, InvitrogenCorporation) and used to treat cells in culture.

D. Lipofectin Encapsulation

20 μg lipofectin (Invitrogen) and 16 μg oligonucleotides were eachincubated with 200 μl Opti-MEM (Invitrogen) media in separate steriletubes at room temperature for 45 min. They were then combined andincubated for an additional 15 min. 1.6 ml Opti-MEM media was then addedto a final volume of 2 ml and a final concentration 1 μMoligonucleotide. The concentration of lipofectin and oligonucleotidescan be adjusted based on their molecular weight and desiredconcentration of compounds. There was no cytotoxic effect at this level.

E. Cell Growth Inhibition Assay

Cell growth inhibition was assessed using3-[4,5-Dimethyl-thiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT)purchased from Sigma Chemical (St. Louis, Mo.). Cells were resuspendedin culture media at 50,000 cells/ml and 100 μl was distributed into eachwell of a 96-well, flat bottomed plate (Costar Corning, N.Y., USA) andincubated for 24 hours. Media was changed to 100 μl fresh mediacontaining the desired concentration of oligonucleotides and incubatedfor 24 hours. Controls had media with pure sterile water equal to thevolume of oligonucleotide solution. The media was changed withoutfurther addition of oligonucleotides every 24 hours until the controlcultures were confluent (6 to 7 days). Thereafter the media was removedand plates were washed two times with phosphate-buffered saline (PBS)and 100 μl of serum free media containing 0.5 mg/ml MTT dye was addedinto each well and incubated for 1 hour at 37° C. The media with dye wasremoved, washed with PBS and 100 μl of dimethyl sulfoxide (DMSO) wasadded to solubilize the reactive dye. The absorbance values were readusing an automatic multiwell spectrophotometer (Bio-Tek MicroplateAutoreader, Winooski, Vt., USA). Each treatment was repeated at least 3times with 8 independent wells each time.

F. Protein Extraction and Western Blot Analysis

The cells were seeded and cultured in T25 tissue culture flasks (Costar,Corning, N.Y., USA) at 200,000 cells/flask. The cells were allowed toattach for 24 hours. The media was replaced with fresh media containing10 to 20 μM oligonucleotides and incubated for 24 hours. The media waschanged every 48 hours without further addition of inhibitors and cellcultures were continued until the control flasks were confluent (6-7days). Cells were harvested using 1× trypsin:EDTA (Invitrogen, Gibco,Md.) and collected by centrifugation at 2000 rpm for 5 min. Cells wereresuspended in 125 mM Tris-HCL buffer (pH 6.8), sonicated with 10-20%output and lysed in an equal volume of 8% SDS for a final concentrationof 4% SDS. Cells extracts were boiled for 10 min, chilled on ice andcentrifuged at 2,000 rpm for 5 min before collecting the supernatant.The protein was quantitated using BCA protein assay kit (Pierce,Rockford, Ill.). 50 to 100 μg of proteins were subjected to 10 to 15%gel (depending on molecular weight of each protein) electrophoresis andtransferred to nitrocellulose membrane (Schleicher & Schuell, Kence,N.H.). Each membrane was blocked with 10% dry milk in TBSTe (Trisbuffered saline, Tween 20) for 2 hr, prior to incubation with primaryantibodies in TBST overnight. Antibodies to human c-myc, c-ha-ras anderbB-2 were mice IgG (Pharmingen San Diego, Calif.). Membranes werewashed 3 times, 15 min each in TBST, then incubated with secondaryantibodies conjugated with peroxidase for 1 hr. The membranes werewashed 5 times, 10 min each in TBST and incubated with 2 ml each ofLumino/Enhancer and Stable peroxide solution (PIERCE) for 1 min. Themembranes were exposed to X-ray film for 2 min (exposure time isadjusted from 10 seconds up to 24 hr if necessary).

Example 2

c-ki-RAS

This example describes the ability of oligonucleotide compounds targetedagainst the promoter of the c-ki-Ras gene to inhibit the growth ofcancer cell lines. Experiments were performed as described in Example 1.The results are shown in FIGS. 13 and 19. The sequences of theoligonucleotides targeted against c-ki-Ras as well as the sequence ofc-ki-Ras gene are shown in FIGS. 5 and 6.

Example 3

Bcl-2

This example describes the ability of oligonucleotide compounds targetedagainst the promoter of the bcl-2 gene to inhibit the growth of cancercell lines. Experiments were performed as described in Example 1. Theresults are shown in FIGS. 14 and 20. The sequences of theoligonucleotides targeted against bcl-2 as well as the sequence of bcl-2gene are shown in FIGS. 1 and 2.

Example 4

c-ha-RAS

This example describes the ability of oligonucleotide compounds targetedagainst the promoter of the c-ha-Ras gene to inhibit the growth ofcancer cell lines. Experiments were performed as described in Example 1.The results are shown in FIGS. 16 and 22. The sequences of theoligonucleotides targeted against c-ha-Ras as well as the sequence ofc-ha-Ras gene are shown in FIGS. 7 and 8.

Example 5

c-erbB-2

This example describes the ability of oligonucleotide compounds targetedagainst the promoter of the c-erbB-2 gene to inhibit the growth ofcancer cell lines. Experiments were performed as described in Example 1.The results are shown in FIGS. 15 and 21. The sequences of theoligonucleotides targeted against c-erbB-2 as well as the sequence ofc-erbB-2 gene are shown in FIGS. 3 and 4.

Example 6

c-myc

This example describes the ability of oligonucleotide compounds targetedagainst the promoter of the c-myc gene to inhibit the growth of cancercell lines. Experiments were performed as described in Example 1. Theresults are shown in FIGS. 17 and 23. The sequences of theoligonucleotides targeted against c-myc as well as the sequence of c-mycgene are shown in FIGS. 9 and 10.

Example 7

TGF-α

This example describes the ability of oligonucleotide compounds targetedagainst the promoter of the TGF-α gene to inhibit the growth of cancercell lines. Experiments were performed as described in Example 1. Theresults are shown in FIGS. 18 and 24. The sequences of theoligonucleotides targeted against TGF-α as well as the sequence of TGF-αgene are shown in FIGS. 11 and 12.

Example 8

Inhibition of Cell Growth by Non-Methylated Oligonucleotides

This example describes the inhibition of growth of lymphoma cell linesby non-methylated oligonucleotides targeted towards Bcl-2. WSU-FSCCLcells were plated in 24 well plates at 2×10⁵ cells/well at t=−24 hr. Foreach time point to be harvested, triplicate wells were treated at t=0with the oligos at the concentrations indicated. Controls were plated intriplicate. Plates were incubated at 37° C. All cultures were monitoredthrough out the experiment by cell count and viability every 24 hr for 4days using trypan blue stain and hemacytometer.

The MABL2 oligonucleotide is targeted to the promoter region of Bcl-2[5′-CAX GCA XGX GCA TCC CXG CCX GTG-3′]. Pho-Mabl-2 is an unmethylatedversion of MABL-2 [5′-CAC GCA CGC GCA TCC CCG CCC GTG-3′].WSU-FSCCL—derived from human B cell lymphoma (low-grade follicularsmall-cleaved cell lymphoma). The experimental protocol is shown inTable 2. TABLE 2 Target Viability Harvest Group Gene Compound CellsConc. Formulation Assay for Methyl 1 Bcl-2 MABL2 FSCCL 10 μM none n = 3@ 24, n = 3 @ 72 48 & 72 hr hr 2 Bcl-2 MABL2 FSCCL  3 μM none n = 3 @24, n = 3 @ 72 48 & 72 hr hr 3 Bcl-2 PhoMABL2 FSCCL 10 μM none n = 3 @24, n = 3 @ 72 48 & 72 hr hr 4 none none FSCCL n/a none n = 3 @ 24, n =3 @ 72 48 & 72 hr hr

The results are shown in FIG. 31. The results demonstrate that theunmethylated oligonucleotide directed against Bcl-2 is as effective asthe methylated oligonucleotide in inhibiting cell growth.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in therelevant fields are intended to be within the scope of the followingclaims.

1. A composition comprising a first oligonucleotide that hybridizesunder physiological conditions to the promoter region of a bcl-2 gene.2. The composition of claim 1, wherein said first oligonucleotide isselected from the group consisting of SEQ ID NOs: 3, 7, 8 and
 9. 3. Thecomposition of claim 1, wherein at least one of the cytosine bases insaid first oligonucleotide is 5-methylcytosine.
 4. The composition ofclaim 3, wherein all of said cytosine bases in said firstoligonucleotide are 5-methylcytosine.
 5. The composition of claim 1,wherein said hybridization of said first oligonucleotide to saidpromoter region of a bcl-2 gene inhibits expression of said bcl-2 gene.6. The composition of claim 1, wherein said bcl-2 gene is on achromosome of a cell, and wherein said hybridization of said firstoligonucleotide to said promoter region of bcl-2 gene reducesproliferation of said cell.
 7. The composition of claim 1, furthercomprising a second oligonucleotide.
 8. The composition of claim 7,wherein at least one of the cytosine bases in said CG dinucleotide pairis 5-methylcytosine.
 9. The composition of claim 7, wherein said secondoligonucleotide is selected from the group consisting of SEQ ID NOs: 3,7, 8 and 9, and wherein said second oligonucleotide is different thansaid first oligonucleotide.
 10. The composition of claim 7, wherein saidsecond oligonucleotide hybridizes to a promoter region of a second gene,wherein said second gene is not bcl-2.
 11. The composition of claim 10,wherein said second gene is an oncogene.
 12. The composition of claim11, wherein said oncogene is selected from the group consisting ofc-ki-Ras, c-Ha-Ras, c-myc, Her-2, and TGF-α.
 13. A compositioncomprising an oligonucleotide that hybridizes to a promoter region of abcl-2 gene at a position selected from the group consisting of betweennucleotides 1-40 of SEQ ID NO:1, between nucleotides 161-350 of SEQ IDNO:1, between nucleotides 401-590 of SEQ ID NO:1 and between nucleotides1002-1260 of SEQ ID NO:
 1. 14. A method, comprising a) providing i) anoligonucleotide selected from the group consisting of SEQ ID NOs: 3, 7,8, and 9; and ii) a cell capable of expressing a bcl-2 gene, and whereinsaid cell is capable of proliferation; and b) introducing saidoligonucleotide to said cell.
 15. The method of claim 14, wherein saidadministering results in a reduction of proliferation of said cell. 16.The method of claim 14, wherein said administration results ininhibition of expression of said bcl-2 gene.
 17. The method of claim 14,wherein said cell is a cancer cell.
 18. The method of claim 14, whereinsaid cell is in a host animal.
 19. The method of claim 18, wherein saidhost animal is a non-human mammal.
 20. The method of claim 18, whereinsaid host animal is a human.
 21. The method of claim 18, wherein saidoligonucleotide is introduced to said host animal at a dosage of between0.01 μg to 100 g.
 22. The method of claim 18, wherein saidoligonucleotide is introduced to said host animal at a dosage of between1 mg to 100 mg per kg of body weight.
 23. The method of claim 18,wherein said oligonucleotide is introduced to said host animal one ormore times per day.
 24. The method of claim 18, wherein saidoligonucleotide is introduced to said host animal continuously.
 25. Themethod of claim 14, wherein said cell is in cell culture.
 26. The methodof claim 14, further comprising the step of introducing a test compoundto said cell.
 27. The method of claim 27, wherein said test compound isa known chemotherapy agent.
 28. The method of claim 17, wherein saidcancer is selected from the group consisting of pancreatic cancer, coloncancer, breast cancer, bladder cancer, lung cancer, leukemia, prostate,lymphoma, ovarian, and melanoma.
 29. The method of claim 19, furthercomprising providing a drug delivery system for introducing saidoligonucleotide to said cell.
 30. The method of claim 29, wherein saiddrug delivery system comprises a liposome, said liposome selected fromthe group consisting of a neutral lipid and a lipid like compound. 31.The method of claim 29, wherein said drug delivery system comprises acell targeting component.
 32. The method of claim 31, wherein said celltargeting compound is selected from the group consisting of a ligand fora cell surface receptor and a ligand for a nuclear receptor.
 33. Themethod of claim 29, wherein said drug delivery system is for use invivo, and wherein oligonucleotide and said liposome are present in theratio of from 2:1 to 1:3/1 μg to 100 mg per kg body weight.
 34. Amethod, comprising a) providing i) an oligonucleotide that hybridizes tothe promoter region of a bcl-2 gene; and ii) a cell comprising a bcl-2gene; and b) introducing said oligonucleotide to said cell.
 35. Themethod of claim 34, wherein said oligonucleotide that hybridizes to saidpromoter region of said bcl-2 gene at a position selected from the groupconsisting of between nucleotides 1-40 of SEQ ID NO:1, betweennucleotides 161-350 of SEQ ID NO:1, between nucleotides 401-590 of SEQID NO:1 and between nucleotides 1002-1260 of SEQ ID NO:
 1. 36. Themethod of claim 34, wherein said oligonucleotide is between 15 and 30bases in length.
 37. The method of claim 34, wherein said at least oneof the cytosine bases in said oligonucleotide is 5-methylcytosine. 38.The method of claim 34, wherein all of said cytosine bases in saidoligonucleotide are 5-methylcytosine.